Subtoxic product levels limit the epoxidation capacity of recombinant E. coli by increasing microbial energy demands.

The utilization of the cellular metabolism for cofactor regeneration is a common motivation for the application of whole cells in redox biocatalysis. Introduction of an active oxidoreductase into a microorganism has profound consequences on metabolism, potentially affecting metabolic and biotransformation efficiency. An ambitious goal of systems biotechnology is to design process-relevant and knowledge-based engineering strategies to improve biocatalyst performance. Metabolic flux analysis (MFA) has shown that the competition for NAD(P)H between redox biocatalysis and the energy metabolism becomes critical during asymmetric styrene epoxidation catalyzed by growing Escherichia coli containing recombinant styrene monooxygenase. Engineering TCA-cycle regulation allowed increased TCA-cycle activities, a delay of acetate formation, and enhanced NAD(P)H yields during batch cultivation. However, at low biomass and product concentrations, the cellular metabolism of both the mutants as well as the native host strains could cope with increased NADH demands during continuous two-liquid phase biotransformations, whereas elevated but still subtoxic product concentrations were found to cause a significantly increased NAD(P)H demand and a compromised efficiency of metabolic operation. In conclusion, operational conditions determine cellular energy and NAD(P)H demands and thus the biocatalytic efficiency of whole-cell redox biocatalysts.

Picoliter nDEP traps enable time-resolved contactless single bacterial cell analysis in controlled microenvironments.

We present a lab-on-a-chip device, the Envirostat 2.0, which allows for the first time contactless cultivation of a single bacterial cell by negative dielectrophoresis (nDEP) in a precisely controllable microenvironment. Stable trapping in perfusing growth medium was achieved by a miniaturization of octupole electrode geometries, matching the dimensions of bacteria. Temperature sensitive fluorescent measurements showed that these reductions of microelectrode distances led to reduced Joule heating during cell manipulation. The presented miniaturization is not possible with conventional manufacturing processes. Therefore, we present a novel bonding technology, which permits miniaturization of 3D octupole electrode geometry with biocompatible materials. To exclude the influence of other cells and to enable sampling of perfusion medium from the isolated living bacterium under study, computer aided flow simulations were used to develop a microfluidic nDEP isolation procedure. The developed microchannel and microelectrode design integrates for the first time well characterized nDEP cell sorting mechanisms and time-resolved contactless single bacterial cell cultivation in a 1.7 picoliter bioreactor system. The cell type independent trapping is demonstrated with singularized Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum and other industrially relevant microbes. The static and precisely controlled microenvironment resulted in a consistent and significant faster growth compared to maximal growth rates observed on population level. Preventing the influence of surfaces and cell-cell interactions, the Envirostat 2.0 chip permits total microenvironmental control by the experimenter and therefore provides major opportunities for microfluidic based cell analysis of bacteria and small eukaryotes.

Single-cell analysis in biotechnology, systems biology, and biocatalysis.

Single-cell analysis (SCA) has been increasingly recognized as the key technology for the elucidation of cellular functions, which are not accessible from bulk measurements on the population level. Thus far, SCA has been achieved by miniaturization of established engineering concepts to match the dimensions of a single cell. However, SCA requires procedures beyond the classical approach of upstream processing, fermentation, and downstream processing because the biological system itself defines the technical demands. This review characterizes currently available microfluidics and microreactors for invasive (i.e., chemical) and noninvasive (i.e., biological) SCA. We describe the recent SCA omics approaches as tools for systems biology and discuss the role of SCA in genomics, transcriptomics, proteomics, metabolomics, and fluxomics. Furthermore, we discuss applications of SCA for biocatalysis and metabolic engineering as well as its potential for bioprocess optimization. Finally, we define present and future challenges for SCA and propose strategies to overcome current limitations.